1. Field of the Invention
The invention concerns devices for the quantitative analysis of chemical substance concentrations in biological fluids and more particularly concerns a disposable device useful for determining the glucose content of biological fluids. The invention also concerns methods of making and using the devices of the invention.
2. Brief Description of the Prior Art
Prior hereto, disposable devices have been known and commercially available which are useful for determining the concentration of glucose (and other substances) in liquids. Of particular importance for their simplicity, low cost, ease of operation and widespread use are the relatively simple devices which comprise a strip of bibulous material or other support element bearing a reagent which yields a color indication upon contact with glucose. Representative of such devices are the devices described in U.S. Pat. Nos. 2,865,718; 2,848,308; 2,893,844; 2,981,606; 3,164,534; 3,212,855; and 3,791,988.
The devices described in the above prior art patents all operate by immersion in e.g. a glucose containing solution for brief periods of time. On contact with glucose, the reagent element of the device undergoes a chemical change, which is indicated visually to the operator by a color change. Generally, the degree of color change is a crude measure of the concentration of glucose in the immersion liquid. For example, the device of U.S. Pat. No. 2,981,606 employs a reagent composition which gives a color indication, depending on the concentration of glucose in the immersion liquid, varying from tints of yellow to green to blue. An approximation of the glucose content of the immersion solution in terms of percentage is then obtained by comparing the color indication with color shades obtained by exposure of the reagent to solutions of known glucose content. In practice, a secondary color comparison chart is used.
Other devices of the prior art employ a single reagent which is sensitive to glucose above a specific concentration. These latter devices, as represented by the devices disclosed in U.S. Pat. No. 2,893,844, also give the operator a visual indication by color formed upon reaction of the reagent element with glucose. The devices do not indicate a range of concentrations, but only indicate if a certain predetermined level of glucose is present or exceeded in the solution being tested.
The disposable devices of the prior art have not offered a means of obtaining a direct numerical reading of glucose or other substance content of the tested solutions. For this determination, either a comparison with a color chart must be made to convert the indicia registered by the device to a numerical value or multiple tests would have to be carried out using a separate indicator device for a number of different minimum glucose level determinations. Both devices offer only a crude approximation of glucose concentrations and are dependent upon the ability of the user of the device to differentiate shades of colors.
A further disadvantage of the commercially available prior art devices resides in the fact that they must be employed following closely defined operating rules. More particularly, the reagents employed continue to react after initial contact with the substance to be detected, such as glucose. The colors formed by the reagents continue to develop to the darkest shade possible and so the indicator must be read within a narrow and specific time period following immersion if any accuracy is to be obtained in the determination. If the operator fails to understand the criticality involved in reading the indicia registered within the prescribed time, or fails to do so accurately, an erroneous reading will be obtained. The indicators of my invention are disposable, register an indicia of dissolved substance concentration which is directly readable in numerical terms and will indicate over any desired range of possible concentrations. In addition, the indicators of my invention provide a registration soon after brief immersion in the containing solution and do not have to be "read" within a critical time period following such immersion. The indicia registrations obtained are stable for reasonably long periods following exposure (particularly to glucose) so that a degree of freedom is obtained as to when the registration must be observed for an accurate reading.
Furthermore, instruments of my making, prepared according to the invention are comparatively insensitive to the effects of interfering substances in the test specimens. In the case of prior art glucose determinations in urine for example, the presence of certain reducing substances such as 2,5-dihydroxy benzoic acid or ascorbic acid in the urine masks the presence of glucose; see for example Feldman et al., Diabetes, Vol. 19, No. 5, Pps. 337-343.
This leads to the creation of potential gross errors in diagnosis and to the erroneous administration or withholding of certain therapeutic drugs. In using the instruments and reagent formulation of my invention however, the effects of such interferences are virtually eliminated, i.e. reduced to about 3% of the effect seen with prior art instruments.
Enzyme based reagent compositions for the determination of glucose in glucose containing solutions known prior to my invention are represented by U.S. Pat. Nos. 2,848,308; 2,981,606; 3,164,534; 3,721,607; and 3,791,988; and the article "Morin et al, Clinical Chemistry, 10/9, 959-952 (1973)"; plus others too numerous to list. Although many of such compositions may be employed in fabricating the indicators of my invention, the preferred indicators employ glucose sensitive reagents which comprise novel compositions of the invention. The novel compositions of the invention, hereinafter described more fully, are advantageously used in the fabrication of the indicators of the invention. The novel compositions are particularly advantageous reagents for the quantitative determination of glucose, being rapid acting and of anticipated enhanced storage stability and result in a reduction of the magnitude of the effect due to interfering substances to about 3% of the effect seen in the use of prior art devices and compositions when used in an instrument of the invention as discussed above. Although the ratio of equivalent proportions of reducing or antagonist compound to equivalent proportion of glucose to be detected may vary within a range of from about 1:1 to 5:1, it is preferred that the ratio be substantially stoichiometric. By stoichiometric I means those amounts which are substantially in the same proportion as the chemically equivalent weight for the reacting species. When my compositions are used, this ratio is essentially stoichiometric, i.e. and therefore predictable and easily controlled in manufacture. Furthermore, only when my preferred compositions are used do I observe the relative absence of interference from reducing substances that may occur in urine and which mask large amounts of glucose, in the case of prior art devices (see above).